Herbal formula PM012 induces neuroprotection in stroke brain

Stroke is a major cause of long-term disability world-wide. Limited pharmacological therapy has been used in stroke patients. Previous studies indicated that herb formula PM012 is neuroprotective against neurotoxin trimethyltin in rat brain, and improved learning and memory in animal models of Alzheimer’s disease. Its action in stroke has not been reported. This study aims to determine PM012-mediated neural protection in cellular and animal models of stroke. Glutamate-mediated neuronal loss and apoptosis were examined in rat primary cortical neuronal cultures. Cultured cells were overexpressed with a Ca++ probe (gCaMP5) by AAV1 and were used to examine Ca++ influx (Ca++i). Adult rats received PM012 before transient middle cerebral artery occlusion (MCAo). Brain tissues were collected for infarction and qRTPCR analysis. In rat primary cortical neuronal cultures, PM012 significantly antagonized glutamate-mediated TUNEL and neuronal loss, as well as NMDA-mediated Ca++i. PM012 significantly reduced brain infarction and improved locomotor activity in stroke rats. PM012 attenuated the expression of IBA1, IL6, and CD86, while upregulated CD206 in the infarcted cortex. ATF6, Bip, CHOP, IRE1, and PERK were significantly down-regulated by PM012. Using HPLC, two potential bioactive molecules, paeoniflorin and 5-hydroxymethylfurfural, were identified in the PM012 extract. Taken together, our data suggest that PM012 is neuroprotective against stroke. The mechanisms of action involve inhibition of Ca++i, inflammation, and apoptosis.

Ischemic stroke accounts for 87% of stroke cases [9] and is a leading cause of adult disability world-wide [10]. Ischemic insult triggers a series of progressive neurodegeneration processes, including glutamate overflow, apoptosis, inflammation, and ER stress, which lead to cell death. Suppressing these processes prevents stroke-mediated degeneration. For example, we previously reported that 2-fucosyllactose reduced ischemia-mediated ER stress and inflammation in brain and improved locomotor behavior in stroke rats [11]. Interestingly, several active ingredients in PM012 also process anti-inflammatory or anti-ER stress properties [12,13]. PM012 may reduce ischemic brain damage through these actions.
The purpose of this study is to examine the protective actions of PM012 in cellular and rat models of stroke. Here we reported that PM012 reduced glutamate-mediated neuronal cell loss and apoptosis in primary neuronal culture. In addition, PM012 improved behavioral function and reduced brain infarction, inflammation, and ER stress in stroke rats. Our data support the notion that PM012 protects against ischemic stroke-mediated neurodegeneration.

Major components in PM012-determined by HPLC
PM012 (650 mg) was dissolved in methanol (5ml) and filtered. The contents in the solution were examined by HPLC. As seen in the chromatograms (Fig 1), two peaks were found at 254 nm and 280 nm, corresponding to paeoniflorin and 5-Hydroxymethylfurfural (5-HMF).

Pretreatment with PM012 reduced brain infarct in stroke rats
A total of 14 stroke rats (vehicle, n = 7, PM012, n = 7) were used for brain infarction analysis. Brain tissues were collected and sectioned into 2 mm slices 2 days after MCAo. Brain

PM012 altered the expression of inflammation-related genes in stroke brains
Cortical tissues from the ischemic side (infarcted) and contralateral side (non-infarcted) hemispheres were collected from 14 stroke rats (veh, n = 7; PM012, n = 7) on day 2. The

PM012 suppressed the expression of ER stress and apoptotic genes in stroke brains
ER stress and apoptotic markers were examined by qRTPCR. The expression of ER stress (ATF6, BIP, CHOP, IRE1, PERK) and apoptotic (Caspase 3) genes were all significantly upregulated in ischemic side cortices (Fig 7, p<0.05, 1-way ANOVA). PM012 significantly reduced the expression of ATF6 ( (Fig 7F, p<0.001). PM012 did not alter the expression of these genes in the non-infarcted side cortex.

Discussions
In this study, we demonstrated that PM012 reduced glutamate-mediated neurotoxicity, apoptosis, and Ca ++ influx in primary neuronal culture in vitro. In addition, pretreatment with PM012 reduced brain infarction, motor deficits, inflammation, and ER stress in stroke brain.
The major finding of this study is that PM012 is neuroprotective against stroke. After the ischemic injury, oxygen-rich blood supplied to the brain is reduced or blocked. Consequently, the reduction of ATP level triggers the influx of calcium ions into the pre-synaptic neurons, and results in glutamate overflow in the synapse and neurotoxicity. Using the endogenous Ca++ probe GCaMP5, we demonstrated that PM012 antagonized NMDA-mediated Ca++i in neurons. Moreover, PM012 reduced glutamate-mediated TUNEL and the loss of MAP2-ir in the neuronal culture, suggesting that PM012 induces neuroprotection through modulation of Ca++i in primary neurons.
We further examined the protective effect of PM012 in vivo. We demonstrated that pretreatment of PM012 for 2 days enhanced locomotor movements and reduced brain infarction in ischemic stroke rats, suggesting that PM012 is also neuroprotective against stroke in vivo. Using HPLC, we identified two molecules, paeoniflorin and 5-HMF, in the PM012 extract. Previous studies have shown that 5-HMF antagonized d-galactosamine and TNF-alpha -mediated ER stress and apoptosis in cultured human hepatocytes [13]. Paeoniflorin prevented lipopolysaccharide-induced overproduction of inflammatory marker IL6 and ER stress markers CHOP and GRP78 in human umbilical vein endothelial cells [12] and attenuated all-trans-retinal-induced ER stress in retinal pigment epithelial cells [15].
Inflammation and ER stress are major causes of neurodegeneration after ischemic brain injury. We and others reported that suppression of inflammation or ER stress improved neural functions in stroke animals [11,16]. These data suggest that paeoniflorin and 5-hydroxymethylfurfural are the active ingredients in PM012, which may induce protection against ischemic brain injury through anti-inflammation and anti-ER stress.
To identify the anti-inflammatory role of PM012 in stroke, we examined the expression of microglia markers in brain. Microglia play an important role in controlling the immune and inflammatory responses after ischemic brain injuries [17]. Two phenotypes (M1 and M2) of microglia with differential actions have been reported [18]. The activation of M1 microglia leads to cell death, while stimulation of M2 microglia increases cell survival [19][20][21]. We demonstrated that PM012 reduced the expression of IBA1, down-regulated M1 markers CD86 and IL6 [22,23], and upregulated M2 marker CD206 [24,25]. Our data suggest that PM012 reduced ischemic brain degeneration by modulating M1/M2 microglia in the infarcted brain.
Similar to previous studies, we demonstrated that ATF6, Bip, CHOP, IRE1, PERK, and Caspase-3 were upregulated after ischemic brain injury. PM012 significantly reduced these ER stress responses in stroke brain. Our findings were further supported by the anti-ER stress effects of two major ingredients (i.e., 5-HMF and Paeoniflorin, see Fig 1) in PM012 [12, 13,  15]. Altogether, our data suggest that PM012 reduced ischemic brain injury through the suppression of ER stress.
In conclusions, PM012 reduced neuronal degeneration, activation of microglia, ER stress, apoptosis, and cerebral infarction in stroke brain. We demonstrated that pretreatment with PM012 reduced ischemic brain damage in rats. For clinical implication, this approach (pretreatment with PM012) may be beneficial to patients with a high risk of stroke (i.e., transient ischemic attack) to prevent recurrent ischemic injury. In our future experiments, we will examine the protective effect of PM012 when given early after stroke.

High-Performance Liquid Chromatography (HPLC) analysis
Chromatographic analysis was performed using a LC-4000 HPLC system (JASCO, Japan) with XTerra TM RP 18 (4.6 x 150 mm, 5 μL) column (Waters, USA). The mobile phase using gradient elution consist of two solvent systems, acetic acid in water (A) and acetic acid in acetonitrile (B). The flow-rate was 1.0 ml/min and injection volume was 10 μg. The column temperature was set at 25˚C. 5-HMF (0.1mg/ml) and paeoniflorin (0.1mg/ml) were dissolved in methanol and used as standards for the qualitative and quantitative analysis of PM012 (2mg/ml). Sample injection volume was 10 μL. Absorbance of column eluate was monitored with a UV spectrometer for 5-HMF at a wavelength of 280 nm and for paeoniflorin at a wavelength of 254 nm.

Primary rat Cortical Neuron (PCN)
Primary cultures were prepared from embryonic (E14-15) cortex tissues obtained from fetuses of timed pregnant rats. The olfactory bulbs, striatum, and hippocampus were removed aseptically, and cortices were dissected. After removing the blood vessels and meninges, pooled cortices were trypsinized (0.05%) for 20 min at room temperature. After rinsing off trypsin with prewarmed Dulbecco's modified Eagle's medium, cells were dissociated by trituration, counted, and plated into 96-well (5.0 × 10 4 / well) cell culture plates pre-coated with polyethyleneimine. The culture plating medium consisted of Neurobasal Medium supplemented with 2% heat-inactivated fetal bovine serum (FBS), 0.5 mM L-glutamine, 0.025-mM L-glutamate, and 2% B27. Cultures were maintained at 37˚C in a humidified atmosphere of 5% CO2 and 95% air. The cultures were fed by exchanging 50% of media with feed media (Neurobasal Medium) with 0.5 mM L-glutamate and 2% B27 with antioxidants supplement on days in vitro (DIV) 3 and 5. PM012 (20 mg) was dissolved in saline (1 mL) and filtered. The solution was further diluted in culture media to various concentrations and added to the culture wells.

Real-time intracellular Ca++ measurement in primary neuronal culture
Primary cortical neuronal cultures were infected by AAV-GCaMP5 (3 × 10 11 viral genome/ ml) on DIV 5 for 1 h. NMDA-mediated Ca ++ influx was examined on DIV12 as previously described [14]. In brief, culture plates were placed on a motorized stage (Prior Scientific Inc., Fulbourn, Cambridge, UK) of a Nikon TE2000 inverted microscope (Nikon, Melville, NY, USA). Microscopic images were recorded through a FITC filter from 1 min before to 7 min after drug treatment at a rate of two frames per sec. The intensity of intracellular green fluorescence of single cells was individually measured by the NIS-Elements AR 3.2 Software (Nikon, Melville, NY, USA).

Immunocytochemistry
Cultured cells were fixed with PFA for 1 h and then washed with PBS. Cells were incubated for 1 day at 4˚C with a mouse monoclonal antibody against MAP2 (1:500) and then rinsed three times with PBS. The bound primary antibody was visualized using Alexa Fluor 488 goat antimouse secondary. Images were acquired using a monochrome camera Qi1-mc attached to a Nikon TE2000-E inverted microscope as previously described [14].

In vitro Terminal Deoxynucleotidyl Transferase (TdT) -Mediated dNTP Nick End Labeling (TUNEL)
Cultures were assayed for DNA fragmentation using a TUNEL -based method as described by the manufacturer (In Situ Cell Death Detection Kit; Roche, Indianapolis, IN). Briefly, 4% PFA fixed cells were permeabilized in 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice. To label damaged nuclei, 50 μL of the TUNEL reaction mixture was added to each sample and kept at 37˚C in a humidified chamber for 60 min. Controls consisted of not adding the label solution (terminal deoxynucleotidyl transferase) to the TUNEL reaction mixture.
The material was examined using a Nikon TE2000 inverted microscope equipped with fluorescence. TUNEL (+) cells were manually counted in 20× images (4 fields per well of 96-well plate).

Animal surgery and drug administration
Rats were anesthetized with sodium pentobarbital (35 mg/kg, i.p.). A craniotomy of about 2-4 mm was made in the right squamosal bone. MCAo was induced by ligating the right distal MCA with a 10-0 suture using methods previously described [26]. After 60 min, the suture on the MCA and arterial clips on common carotids were removed to allow reperfusion. Core body temperature was monitored and maintained at 37˚C. After recovery from anesthesia, body temperature was maintained at 37˚C using a temperature-controlled incubator. Control animals received sham surgery, including craniotomy without MCAo. PM012 (50 mg) was added to saline (1 mL) and then vortexed for 1 min. PM012 (50 mg/kg/d x 2) or vehicle (saline) was given intraperinoneally from 2 days before the MCAo.

Locomotor activity measurements
Animals were individually placed in 42 × 42 × 31 cm open plexiglass boxes. Locomotor activity was recorded with an infra-red activity monitor (Accuscan, Columbus, OH) for 2 h (12-h light and 12-h dark/day) on day 2 (pretreatment experiment) after the MCAo [27]. The monitor contained eight vertical infrared sensors situated 10 cm from the floor of the chamber. Motor activities were calculated by the number of beams broken for 2 h after placement in the chamber. Vertical activity (VACTV; the total number of beam interruptions that occurred in the vertical sensors), total distance traveled (TOTDIST; the distance traveled in centimeters), and horizontal activity (HACTV; the total number of beam interruptions that occurred in the horizontal sensor) were analyzed by the Versamax program (Accuscan, Columbus, OH).

2,3,5-Triphenyltetrazolium Chloride (TTC) staining
Rats were decapitated 2 days after MCAo. The brains were removed and sliced into 2.0-mm sections. The brain slices were incubated in 2% TTC solution for 5 min at room temperature and then transferred into a 4% PFA solution for fixation. The area of infarction in each slice was measured with a digital scanner and the Image Tools program (University of Texas Health Sciences Center).

Quantitative Reverse Transcription PCR (qRTPCR)
Cortical tissues from the infarcted and non-infarcted hemispheres were collected. Total RNAs were isolated using TRIzol Reagent (ThermoFisher, #15596-018, Waltham, MA, USA), and cDNAs were synthesized from 1 μg of total RNA by use of a RevertAid H Minus First-Strand cDNA Synthesis Kit (Thermo Scientific, #K1631, Waltham, MA, USA). cDNA levels for CD86, CD206, IBA1, IL-6, PERK, IRE1, CHOP, BIP, ATF6, caspase3, actin, and GAPDH were determined using specific universal probe library primer-probe sets or gene-specific primers (Table 1). Samples were mixed with TaqMan Fast Advanced Master Mix (Life Technologies, #4444557, Carlsbad, CA, USA) or SYBR (Luminaris Color HiGreen Low ROX qPCR Master Mix; ThermoScientific, Waltham, MA, USA). Quantitative realtime PCR (qRT-PCR) was carried out using the QuantStudio™ 3 Real-Time PCR System (Thermo Scientific,Waltham, MA, USA). The expression of the target genes was normalized relative to the endogenous reference genes (beta-actin and GAPDH averages) using a modified delta-delta-Ct algorithm. All experiments were carried out in duplicate.

Statistics
Data are presented as the mean ± SEM. Unpaired t-test, one-or two-way ANOVA, and posthoc Newman-Keuls (NK) test were used for statistical comparisons, with a significance level of p < 0.05.